This invention relates to a rapid diagnostic test for the presence of Bluetongue vital specific antibody in serum. More particularly, the invention relates to a modified competitive enzyme linked immunosorbent assay for the detection in serum of the presence of Bluetongue viral specific antibody.
Bluetongue is an insect-borne disease of domestic (sheep, goats, cattle, water-buffaloes, camels, etc.) and wild (blesbok, white-tailed deer, elk, bighorn sheep, mule deer, pronghorn antelope, etc.) ruminants. In sheep and wild ruminants the disease is acute and the mortality is often high, whereas in cattle and goats the disease is usually milder.
First discovered a century ago and originally thought to be confined to Africa, confirmed outbreaks of Bluetongue virus have been reported in Europe, North and Latin America, and in the Near and Far East (Ozawa, Y., "Overview of the World Situation" in Bluetongue and Related Orbiviruses, Alan Liss, Inc., 1985, pages 13 to 20). Recent surveys indicate that Bluetongue virus is spreading silently to almost all the tropical and sub-tropical countries of the world (ibid., page 16).
In sheep the disease is characterized by fever, emaciation, oral lesions (often involving inflammation, ulceration, and necrosis of the tongue, lips and dental pads), and lameness (Callis, J. J., et al., Illustrated Manual for the Recognition and Diagnosis of Certain Animal Diseases, Mexico-United States Commission for the Prevention of Foot and Mouth Disease, 1982, pages 56 to 61). Focal hemorrhage may be present on the lips and gums, and the tongue may become edematous and congested or cyanotic, giving the disease its name. Morbidity is 80-100% in fully susceptible sheep; mortality is variable, from 0 to 50% (ibid., page 57).
Bluetongue is caused by a member of the genus Orbivirus (Reoviridae), commonly called Bluetongue virus or BTV. BTV is primarily transmitted by biting midges of the genus Culicoides. Infected cattle, which experience a prolonged viremia in contrast to sheep, provide a reservoir for the dissemination of the disease (Roy, P., 13 Virus Research 179-206 (1989)). Inapparent or latent infection may persist in cattle for a number of years, and recrudescence of the disease may be stimulated by the bites of Culicoides. Venereal transmission from an infected bull to a bred cow has also been demonstrated (Jubb, K. V. F., et al., Pathology of Domestic Animals, vol. 2, Academic Press, 1985, pages 108 to 112).
The diagnosis of Bluetongue generally involves either the isolation and identification of the virus or the detection of viral antigen-specific antibodies in serum (Jochim, M. M., "An Overview of Diagnostics for Bluetongue," Bluetongue and Related Orbiviruses, Alan Liss, Inc., 1985, pages 423 to 433). An example of the former type is described in U.S. Pat. No. 4,873,189 to Jochim and Jones (column 4, lines 42 to 59); virus from an infected animal isolated and adapted to grow in cell culture is complexed with BTV monoclonal antibody and identified in an indirect fluorescent antibody test. However, since serologic tests are easier and more economical to perform, they have been used more extensively than virus isolation to study the epizootiology of the disease.
The serology of Bluetongue is complicated by the identification of 24 different virus serotypes from different parts of the world, including five in North America (denoted serotypes 2, 10, 11, 13, and 17; see Roy, P., cited above, page 180). Although these may represent not so much distinct types as points on a spectrum of antigenicity brought about by reassortment of segmented orbivirus genomes (and, in fact, sequence analyses of BTV genomes have confirmed that all the genes representing the non-structural proteins of the virus as well as most of the inner capsid polypeptides are highly conserved, whereas the outer capsid polypeptides vary considerably; see Roy, P., cited above, at page 201), immunity to one serotype does not confer resistance against another and may cause "sensitization," with a more severe syndrome following infection by a second type (Judd, K. V. F., cited above, page 110). Moreover, epizootic hemorrhagic disease virus (EHDV), another orbivirus also spread by Culicoides midges (sometimes at the same time), is serologically related to BTV, and immunological cross-reactions between BTV and EHDV have been seen with a number of virus-specified proteins. Serological cross-reactions can hinder accurate diagnosis of BTV.
Serologic tests that have been developed to assay for antibodies have been recently reviewed (see the Jochim, M. M., chapter cited above, pages 427 to 431). These include complement fixation, immunodiffusion, immunofluorescence, hemolysis-in-gel and enzyme linked immunosorbent assay (ELISA). An agar gel immunodiffusion test is most widely used for detection of group-specific anti-BTV antibodies (Afshar, A., et al., J. Clin. Microbiol. 25:1705-1710 (1987)). However, the test is not highly sensitive, specific, or quantitative, and interpretation of the results is often subjective.
As an alternative, several ELISAs have been developed. The first suggested was an indirect (or I-) ELISA (see Afshar, et al., cited above, and Lunt, R. A., et al., J. Gen. Virol. 69:2729-2740 (1988)). In this procedure, multiple wells on microtiter plates are coated with BTV antigen by passive adsorption for 18 hours, then washed and incubated with test sera for 3 hours, then washed and incubated with horseradish peroxidase (HRPO) labelled with antibovine IgG or antiovine IgG for an hour, and then incubated with an HRPO chromogenic substrate and hydrogen peroxide (H.sub.2 O.sub.2) for 30 minutes; the appearance of color (read as an optical density value at a wavelength appropriate for the HRPO-oxidized chromogen) is read as a negative result (Afshar, et al. cited above, page 1706).
The test was described as being reliable, but it failed to detect early postinfection reactions of experimentally infected animals (ibid.). Moreover, problems with cross-reactivity with other orbiviruses were encountered (Lunt, et al., cited above). To circumvent these problems, a competitive (or c-) ELISA was developed. This method was similar to the I-ELISA except that test serum was incubated with monoclonal antibody for 3 hours in the second step; the IgG labelled-HRPO and HRPO substrate incubations were the same (Afshar, et al., cited above, page 1706).
This test was subsequently modified in a blocking (B-) ELISA, which replaced the three-hour serum/antibody/antigen incubation (second step) with three one-hour incubations: an incubation of serum with antigen followed by an incubation with added monoclonal antibody followed by an incubation with added IgG labelled-HRPO (Lunt, et al., cited above).
Cross-reactivity with other orbiviruses continued to plague the results, however. Animals which had undergone separate infections with both BTV and EHVD and animals from regions of high orbivirus activity exhibited anomalous titers (ibid.). Moreover, no assessment was made of immunological responses from mouse-derived components inherent in a procedure involving mouse brain-adapted virus, which may have interfered non-specifically in the assay.